Construction and evaluation method of canine spontaneous breast cancer organoid model

Abstract

The invention belongs to the cross technical field of veterinary medicine and biomedicine, and relates to a construction and evaluation method of a dog spontaneous breast cancer organoid model, which specifically comprises the following steps: (1) obtaining dog spontaneous breast cancer tissues through operation or biopsy, separating tumor tissues under sterile conditions, and carrying out fine enzymolysis digestion treatment to obtain dog spontaneous breast cancer tissues; obtaining a single-cell suspension; and (2) inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with a growth factor, promoting the growth and amplification of the breast cancer organs, and carrying out timely passage according to the growth condition so as to maintain the activity of the model. The model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of primary tumors, does not need gene editing, is simple and convenient to operate and low in cost, is particularly suitable for individual drug sensitivity detection of pet dogs, and provides an efficient and high-simulation experimental platform for dog breast cancer mechanism research and clinical precise medication.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary medicine and biomedical cross technology, and relates to a method for constructing and evaluating a canine spontaneous breast cancer organoid model, in particular to a method for constructing a three-dimensional organoid model using canine spontaneous breast cancer tissue, and an evaluation system for evaluating the efficacy and toxicity of anti-tumor drugs through multi-drug gradient. BACKGROUND

[0002] Breast cancer is one of the most common malignant tumors in female dogs, with complex pathogenesis and strong heterogeneity, and limited clinical treatment options. At present, the research and treatment of canine breast cancer are mostly based on human medical achievements, but due to species differences, individual genetic background diversity, and lack of clinical trial data for pets, the effects of drug treatment are often inconsistent, and the toxic side effects are difficult to predict.

[0003] In the prior art, canine tumor research mostly relies on cell lines or transplanted tumor models, which not only cannot preserve the microenvironment structure and cell heterogeneity of the primary tumor, but also are difficult to simulate the dynamic evolution process of the tumor in vivo. For example, traditional two-dimensional cell culture models lack cell-matrix interaction and tumor tissue structure, resulting in a large deviation between drug response and actual situation in vivo; and transplanted tumor models have high cost, long cycle, and great ethical controversy, and are not suitable for high-throughput drug screening.

[0004] Although organoid technology has been widely used in human tumor research, it can better maintain the original characteristics and drug sensitivity of the tumor, but its application in pet medicine, especially in canine spontaneous tumors, is still in its infancy. The culture medium formula of human breast cancer organoids is relatively mature, but canine cells have different needs for the types and concentrations of growth factors, and direct application cannot effectively culture organoids. And successfully culturing spherical structures does not mean functional breast cancer organoids, and identification is necessary to ensure the production of canine breast cancer organoids.

[0005] At present, most organoid models rely on gene editing or exogenous mutation introduction, which not only has complex operation and high cost, but also cannot accurately reflect the mutation spectrum and microenvironment composition of naturally occurring tumors, limiting its value in individualized treatment guidance.

[0006] In addition, the existing canine tumor efficacy evaluation system is mostly limited to cell viability detection, lacking multi-dimensional analysis of tumor markers (such as HER-2, ER), proliferation activity (such as KI-67), and functional proteins (such as pan-Cytokeratin), making it difficult to comprehensively evaluate the mechanism of action and potential toxicity of drugs. More importantly, the pet clinic lacks a standardized and repeatable drug sensitivity detection platform, resulting in that treatment decisions are still mainly based on experience, lacking data support.

[0007] Therefore, developing a high-simulation model capable of truly reflecting the biological characteristics and drug response of canine breast cancer has become an urgent need in the field of pet precision medicine. SUMMARY

[0008] In order to solve the problems of lack of pet tumor model, inaccurate drug efficacy evaluation and lack of individualized treatment data in the prior art, the purpose of the present application is to establish a kind of organoid construction and evaluation system based on canine spontaneous breast cancer tissue, which can: (1) maximize the retention of tumor tissue structure, cell heterogeneity and microenvironment characteristics; (2) establish a drug efficacy evaluation index system covering pathology, molecule, function and other multiple levels; (3) support high-throughput sensitivity test of various chemotherapy drugs and combinations, and provide operable individualized medication basis for clinic.

[0009] In order to achieve the above purpose, The first aspect of the present application discloses a construction method of canine spontaneous breast cancer organoid model, which comprises the following steps: (1) Obtain canine spontaneous breast cancer tissue by surgery or biopsy, and perform fine enzymatic digestion treatment on the tumor tissue under sterile conditions to obtain a single cell suspension; (2) Inoculate the single cell suspension into a three-dimensional culture system, and use a culture medium containing epidermal growth factor (EGF), fibroblast growth factor (FGF) and B27 for three-dimensional culture to promote the growth and expansion of breast cancer organoids; according to the cell growth condition, perform subculture operation to ensure the continuous culture and expansion of the organoids.

[0010] In step (1), the enzymatic digestion is combined treatment of collagenase IV 1 mg / mL and trypsin 0.25%, and the fine enzymatic digestion is performed at 37℃ for 30 minutes. Digestion is a key step: insufficient digestion will produce too large cell clumps, affecting the formation of organoids; and excessive digestion (such as too long enzyme action time or too high concentration) will seriously damage cell activity. Even cause cell death. Through experiments and optimization, the present application determines the best type, concentration and action time of digestion enzymes such as collagenase and trypsin.

[0011] In step (2), the three-dimensional culture system is a three-dimensional culture system supported by Matrigel, and the three-dimensional culture system is supplemented with the following components: Basic additives: 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 2 mM GlutaMAX; Growth factors: 50 ng / mL EGF, 100 ng / mL FGF10; Small molecule inhibitors: 500 nM A83-01, 10 μM Y-27632; Other additives: B27 (1x), 1 mM N-acetyl cysteine.

[0012] Organoid culture medium requires a set of precisely formulated growth factors (such as EGF, FGF, Noggin, R-spondin1, Wnt3a, etc.) to mimic the stem cell microenvironment (Niche) in vivo. The present application develops a specific growth factor formula for canine cells.

[0013] In some embodiments, the passaging operation in step (1) can use mechanical blowing method (such as using 27G needle to blow gently), which can disperse the organoids into small clumps containing 50-100 cells, which helps to maintain the activity of stem cells.

[0014] In some embodiments, the three-dimensional culture system in step (2) can also use Corning® Matrigel, and the culture medium can be combined with commercial organoid culture additives.

[0015] Preferably, for the verification of the canine spontaneous breast cancer organoid model, the following methods can be used: The mature organoids are subjected to the following molecular marker detection and pathological analysis: Immunohistochemistry: detection of pan-Cytokeratin (epithelial marker), Estrogen Receptor (estrogen receptor) and KI-67 (proliferation index); HE staining: observation of cell heterotypic, karyomit and tissue structure, scoring according to breast cancer pathological standard; if necessary, qPCR or sequencing can be used to verify the expression state of specific genes.

[0016] The verification of the canine spontaneous breast cancer organoid model can also combine live cell imaging system to dynamically record the growth of the organoids, or analyze the consistency of the transcriptome between the organoids and the primary tumor (correlation>85%) through single cell RNA sequencing (scRNA-seq).

[0017] The second aspect of the present application discloses an evaluation method of the above-mentioned canine spontaneous breast cancer organoid model, characterized in that the canine spontaneous breast cancer organoid model is evaluated by at least one of the following evaluation methods: 1) Pathological feature evaluation: the microscopic structure of the canine spontaneous breast cancer organoid (such as whether the boundary is infiltrated, whether the cell arrangement is disordered) is observed under a microscope, and the pathological features such as nuclear-cytoplasmic ratio and karyomit are analyzed by HE staining section, and the score is given according to the histological standard of breast cancer. 2) or drug screening evaluation: divide the organoids into control and experimental groups, and give the experimental group irinotecan, etoposide, cisplatin and other drugs (concentration gradient is 0.1-20 μM), and after 72 hours of treatment, the cell survival rate is detected by CCK-8 method, and the drug sensitivity (including the inhibition effect of the drug on tumor growth and the toxicity threshold) is evaluated; In some embodiments, the volume change of the organoids can be dynamically monitored by a real-time fluorescence imaging system (such as IncuCyte), such as a 50% volume reduction after 48 hours of irinotecan (10 μM) treatment; 3) or cell response evaluation: the response degree of the canine spontaneous breast cancer organoid model to the drug is evaluated by detecting the cell survival rate, proliferation ability and apoptosis; it can include PD-L1 immunohistochemical score, such as model group PD-L1 positive rate > 30%, which indicates that it may be sensitive to immunotherapy; 4) or tumor marker detection: the expression changes of human epidermal growth factor receptor 2 (HER-2), vascular endothelial growth factor (VEGFR), carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA15-3) in the breast cancer organoid model are detected by immunofluorescence or ELISA technology; 5) or functional impairment evaluation: the expression of pan-cytokeratin, estrogen receptor and KI-67 protein is analyzed by immunofluorescence staining, and the influence of the drug on the epithelial characteristics, hormone receptor status and proliferation activity of tumor cells is evaluated.

[0018] The pathological feature evaluation further includes recording the adenocarcinoma characteristics by H&E staining section, including cell polarity disorder, nuclear atypia and destruction of gland lumen structure.

[0019] In the cell response evaluation, the cell survival rate at different time points is determined by CCK8 method, and a dose-effect curve is drawn.

[0020] The third aspect of the present application discloses the application of the breast cancer organoid model constructed by the above-mentioned spontaneous breast cancer organoid model construction method in anti-tumor drug screening, toxicity evaluation or individualized treatment scheme making. It includes: 1) drug screening: used for high-throughput screening of chemotherapeutic drugs, targeted drugs (such as anti-HER-2 drugs) and immunotherapy drugs; 2) toxicity evaluation: evaluate the potential toxicity of the drug on normal canine mammary gland organoids; 3) mechanism research: study the breast cancer occurrence, development and drug resistance mechanism, or the interaction between tumor and microenvironment.

[0021] The canine spontaneous breast cancer organoid model used in the above application comprises exposing a cultured mature breast cancer organoid to an anti-tumor drug gradient of irinotecan, etoposide and cisplatin, and constructing a drug response type breast cancer organoid model by adjusting the type, concentration and action time of the drug.

[0022] The drug gradient is irinotecan (1-20 muM), etoposide (1-20 muM), and cisplatin (1-20 muM), and the structural changes, apoptosis and drug response characteristics of the organoid are observed under a microscope, and the effectiveness of the model is verified by combining molecular biology and functional means.

[0023] The fourth aspect of the present application discloses the application of the evaluation method of the above-mentioned canine spontaneous breast cancer organoid model in evaluating the pathological characteristics, drug sensitivity, tumor marker expression and functional damage degree of the breast cancer model. It comprises: 1) Individualized treatment: based on the drug sensitivity test of the canine-derived organoid (Canine-Derived Organoid, CDO), a basis is provided for guiding clinical drug use; 2) Biomarker exploration: screening of molecular markers related to disease prognosis or drug response through proteomics, metabolomics and other omics data.

[0024] Compared with the prior art, the present application has the following advantages: (1) The present application provides a standardized and repeatable canine breast cancer organoid construction and drug sensitivity detection platform for the first time, which makes up for the short board of the lack of reliable in vitro model for pet tumors; (2) The present application develops and designs a culture medium formula suitable for the construction of canine breast cancer organoids according to the cell characteristics of canine organisms, solving the problem of unsuitability of directly using human organoid culture system; (3) The model is directly constructed from canine spontaneous breast cancer tissue, which completely retains the genetic heterogeneity, tissue structure, cell type diversity and microenvironment characteristics of the tumor, does not require exogenous gene editing, and is more close to the biological behavior of naturally occurring tumors; (4) Using the organoid amplification technology of the present application, a single sample can build hundreds of detection units, reducing the cost by 90%, and supporting 96-well plate high-throughput drug screening; (5) Multi-dimensional evaluation system: integrating pathological, functional and molecular marker (such as HER-2, ER, KI-67) analysis, comprehensively evaluating drug response and mechanism; (6) Ethics and expansibility: significantly reducing the use of experimental animals, complying with the 3R principle, and can be expanded to other types of pet tumor models. BRIEF DESCRIPTION OF DRAWINGS

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This image shows a three-dimensional culture of canine breast cancer organoids.

[0027] Figure 2 Immunohistochemical verification of three-dimensional culture of canine breast cancer organoids.

[0028] Figure 3 HE staining pathological assessment of canine breast cancer organoids.

[0029] Figure 4 The drug exhibits a concentration-dependent inhibition of organoid survival.

[0030] Figure 5A -C indicates the expression of key proteins using immunofluorescence staining.

[0031] Figure 6 This demonstrates the detection of tumor marker secretion levels using the ELISA method. Detailed Implementation

[0032] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0033] Example 1 The method for constructing a canine spontaneous breast cancer organoid model includes the following steps: (1) Preparation and culture of breast cancer organoids Clinically diagnosed canine spontaneous mammary carcinoma tissues were selected, and the tumor tissues were separated by aseptic surgery. After removing the necrotic parts, the tissues were finely enzymatically digested at 37°C for 30 minutes with collagenase IV (1 mg / mL) and trypsin (0.25%). The tissues were then centrifuged at 1000 rpm for 5 minutes to obtain a single-cell suspension, which was then filtered through a 40 μm cell sieve to remove cell clumps.

[0034] (2) Establishment of a three-dimensional culture system and organoid expansion Single-cell suspensions were seeded in Matrigel at a density of 5 × 10^4 cells / mL and covered with culture medium containing Advanced DMEM / F12, supplemented with the following components: Growth factors: EGF (50 ng / mL), FGF10 (100 ng / mL); Small molecule inhibitors: A83-01 (500 nM, used to inhibit TGF-β signaling), Y-27632 (10 μM, used to inhibit the ROCK pathway). Basic additives: B27 (1×), N-acetylcysteine ​​(1 mM), GlutaMAX (2 mM).

[0035] Cultured at 37°C and 5% CO2, with the culture medium changed every 3 days, organoids typically form stable structures within 5–7 days. Depending on growth density, passages were performed using Accutase digestion or mechanical pipetting, and re-seeded at a 1:3 ratio to maintain expansion. Results are as follows: Figure 1 As shown, after 7 days of culture, the organoids prepared by the above method (control group) can form typical mammary organoid structures, which are spherical and have clear boundaries. Construction of a drug sensitivity assessment model: Mature breast cancer organoids were exposed to different concentrations of antitumor drugs (including but not limited to irinotecan, etoposide, and cisplatin). Morphological changes in the organoids were observed under a microscope, and cell viability and morphological characteristics were recorded. The effectiveness of the breast cancer organoid model in drug sensitivity assessment was verified by combining molecular biology, immunology, histology, and functional evaluation methods. Results are as follows: Figure 1 As shown in the cisplatin, irinotecan, and etoposide groups, the organoid morphology of the cisplatin, irinotecan, and etoposide groups was significantly altered, with loose structures and blurred boundaries, exhibiting varying degrees of fragmented or dispersed growth, suggesting that cisplatin, irinotecan, and etoposide have an impact on the three-dimensional growth morphology of canine mammary gland organoids.

[0036] Example 2 Experimental methods: Canine spontaneous breast cancer organoid model samples (control group) from Example 1 were taken, paraffin-embedded and sectioned (4μm thick), and immunohistochemical staining was performed to detect the expression of pan-Cytokeratin, Estrogen Receptor and KI-67.

[0037] The results are as follows Figure 2 As shown, immunohistochemical results showed that pan-Cytokeratin (epithelial marker), Estrogen Receptor (hormone receptor), and KI-67 (proliferation marker) were all positively expressed in the organoids, further confirming that the model has typical molecular characteristics of breast cancer.

[0038] Example 3 Experimental methods: Canine spontaneous breast cancer organoid model samples were taken, embedded in paraffin, sectioned (4μm thick), and stained with hematoxylin and eosin (HE).

[0039] Microscope (40x objective) was used to observe cell arrangement, nuclear-cytoplasmic ratio and glandular lumen structure.

[0040] Results: The control group showed typical adenocarcinoma structure, with disordered cell arrangement, obvious nuclear atypia, and pathological mitotic figures, which was consistent with the pathological features of breast cancer. Figure 3 A large number of karyopyknosis cells appeared in the drug treatment groups (cisplatin group, irinotecan group and etoposide group), and the proportion increased significantly, indicating that the drugs successfully induced tumor cell apoptosis. Figure 3

[0041] Example 4 Concentration-dependent inhibition of drug on organoid survival rate Method: CCK-8 method was used to detect the survival rate of organoids after treatment with different concentrations of irinotecan (1-20 μM), etoposide (1-20 μM), and cisplatin (1-20 μM) for 72 hours, and the dose-effect curve was drawn.

[0042] Results: As shown in Figure 4 , cisplatin, irinotecan, and etoposide could significantly inhibit the survival of organoids, and the inhibitory effect was concentration-dependent: as the drug concentration increased in the range of 1-20 μM, the survival rate of organoids gradually decreased; within each drug group, different concentrations (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) were significantly different from the control group with a concentration of 0 (indicated by statistical significance in the figure), indicating that the three drugs had a concentration-dependent inhibitory effect on the survival of canine breast cancer organoids.

[0043] Example 5 Immunofluorescence staining analysis of key protein expression Method: Immunofluorescence staining was performed on the control group and drug treatment groups (cisplatin group, irinotecan group, and etoposide group, with a uniform concentration of 10 uM) to detect the following markers: pan-Cytokeratin (epithelial marker), Estrogen Receptor (estrogen receptor), KI-67 (proliferation index), and cell nucleus (DAPI, blue) Results as shown in Figure 5A -C, pan-Cytokeratin, Estrogen Receptor, and KI-67 were highly expressed in the model group; after irinotecan treatment, the expression of the three markers decreased significantly, indicating that the drug inhibited epithelial phenotype, hormone signaling pathway, and cell proliferation.

[0044] Example 6 ELISA method for detecting tumor marker secretion level ​Methods: The supernatant of organoids culture (10uM concentration) was collected and the concentration of HER-2, VEGFR, CEA and CA15-3 was quantitatively detected by ELISA.

[0045] Results: As shown in Figure 6 Figure 1, the expression of HER-2, VEGFR, CEA and CA15-3 in the supernatant of organoids culture in the control group was at a high level; after treatment with cisplatin, irinotecan and etoposide, the secretion levels of each marker decreased to varying degrees, and there were significant statistical differences (the **, ***, **** marks in the figure represent different levels of significance). Among them, HER-2 and CA15-3 decreased more significantly in the irinotecan treatment group, indicating that irinotecan had a relatively more prominent regulatory effect on these two tumor-related markers; at the same time, VEGFR, CEA and other markers also showed a significant downward trend with drug treatment, indicating that cisplatin, irinotecan and etoposide could all participate in the regulation of the biological processes of canine breast cancer organoids by affecting the secretion of these markers.

[0046] Example 7: Passage and expansion of canine breast cancer organoids: When the diameter of the organoids in Example 1 (control group) reached 100-200 μm, Accutase enzyme digestion (37℃, 10 minutes) or mechanical blowing (27G needle) was used to disperse the organoids into small cell clusters (50-100 cells per cluster).

[0047] The organoids were reseeded in fresh Matrigel at a ratio of 1:3 and maintained under the same culture conditions. The morphology, size and growth rate of the organoids were recorded at each passage to ensure the stability of the culture system. After more than 5 consecutive passages, the expression of pan-Cytokeratin, Estrogen Receptor and KI-67 was detected by immunofluorescence to ensure the consistency of the biological characteristics of the model. The results showed that the organoids prepared by the present application could be continuously passaged.

[0048] The technical solutions and experimental specifications of the tumor model construction method based on canine spontaneous breast cancer organoids and the multi-dimensional evaluation system provided by the present application. These embodiments not only ensure the accuracy and repeatability of experimental data through standardized operation procedures, but also provide a highly simulated experimental platform for anti-tumor drug screening, treatment toxicity evaluation, tumor development mechanism research and individualized treatment plan optimization for dogs. This model integrates pathological, molecular biological and functional evaluation indicators at multiple levels to achieve a comprehensive analysis of the biological characteristics of breast cancer and drug response.

[0049] The above content fully demonstrates the basic principles, technical features and application value of the present application in pet precision medicine.

[0050] Application scenario examples: 1. Pet hospital: based on tumor samples of sick dogs to construct organoids, test chemotherapy drug sensitivity, and develop individualized treatment plans; 2. Pharmaceutical enterprises: preclinical potency and toxicity evaluation of new anti-tumor drugs for dogs; 3. Research institutions: study of dog breast cancer mechanism, drug resistance evolution and tumor-microenvironment interaction.

[0051] It should be noted that the present application is not limited to the implementation described in the above specific examples, and those skilled in the art should understand that these examples are only used to illustrate the core principles of the present application. Without departing from the design idea and protection scope of the present application, those skilled in the art can make appropriate adjustments and improvements to the model construction method, evaluation index and application scene according to actual needs. The protection scope of the present application is subject to the appended claims, and any equivalent replacement or reasonable improvement based on the technical solutions of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a canine spontaneous breast cancer organoid model, characterized in that, The construction method includes the following steps: (1) Spontaneous mammary gland tissue from dogs was obtained by surgery or biopsy, and the tumor tissue was subjected to fine enzymatic digestion under sterile conditions to obtain a single-cell suspension; (2) Single cell suspensions were inoculated into a three-dimensional culture system and cultured in a medium containing epidermal growth factor (EGF), fibroblast growth factor (FGF) and B27 to promote the growth and expansion of breast cancer organoids. According to the cell growth, passage operations were performed to ensure the continuous culture and expansion of organoids.

2. The method for constructing a spontaneous breast cancer organoid model according to claim 1, characterized in that, In step (1), the enzymatic digestion is performed by a combination of collagenase IV 1 mg / mL and trypsin 0.25%.

3. The method for constructing a spontaneous breast cancer organoid model according to claim 1, characterized in that, In step (2), the three-dimensional culture system is a matrix gel-supported three-dimensional culture system, and the three-dimensional culture system is supplemented with the following components: Basic additives: 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 2 mM GlutaMAX; Growth factors: 50 ng / mL EGF, 100 ng / mL FGF10; Small molecule inhibitors: 500 nM A83-01, 10 μM Y-27632; Other additives: B27 (1×), 1 mM N-acetylcysteine.

4. The evaluation method for canine spontaneous breast cancer organoid models constructed by the method described in any one of claims 1-3, characterized in that, Evaluate canine spontaneous breast cancer organoid models using at least one of the following assessment methods: 1) Pathological feature assessment: The pathological features of the canine spontaneous breast cancer organoid model were assessed to determine whether they met expectations by observing the microstructure, cell morphology and adenocarcinoma characteristics. 2) Drug screening and evaluation: Irinotecan, etoposide and cisplatin were used to treat a canine spontaneous breast cancer organoid model to evaluate the inhibitory effect of the drugs on tumor growth and the toxicity threshold. 3) Or cell response assessment: The degree of response of a canine spontaneous breast cancer organoid model to drugs is assessed by detecting cell viability, proliferation capacity and apoptosis. 4) Tumor marker detection: Immunofluorescence or ELISA techniques were used to detect changes in the expression of human epidermal growth factor receptor 2 (HER-2), vascular endothelial growth factor (VEGFR), carcinoembryonic antigen (CEA), and carbohydrate antigen 15-3 (CA15-3) in breast cancer organoid models. 5) Functional impairment assessment: Immunofluorescence staining was used to analyze the expression of pan-Cytokeratin, Estrogen Receptor, and KI-67 proteins to assess the effects of the drug on tumor cell epithelial characteristics, hormone receptor status, and proliferative activity.

5. The evaluation method for a canine spontaneous breast cancer organoid model according to claim 4, characterized in that, The pathological feature assessment also includes recording adenocarcinoma features through H&E stained sections, including cell polarity disorder, nuclear atypia, and destruction of glandular lumen structure.

6. The evaluation method for canine spontaneous breast cancer organoid models according to claim 4, characterized in that, In the cell response assessment, the CCK8 assay was used to determine cell viability at different time points, and dose-response curves were plotted.

7. The application of a breast cancer organoid model constructed based on the method for constructing a canine spontaneous breast cancer organoid model according to any one of claims 1-3 in the screening of antitumor drugs, toxicity assessment or individualized treatment plan formulation.

8. The application according to claim 7, characterized in that, The application involves exposing cultured mature breast cancer organoids to a gradient of antitumor drugs, namely irinotecan, etoposide, and cisplatin, and constructing drug-responsive breast cancer organoid models by adjusting the types, concentrations, and durations of action of the drugs.

9. The application according to claim 8, characterized in that, The drug gradient consisted of irinotecan (1-20 μM), etoposide (1-20 μM), and cisplatin (1-20 μM). The changes in organoid structure, apoptosis, and drug response characteristics were observed under a microscope, and the effectiveness of the model was verified by combining molecular biology and functional methods.

10. An evaluation method based on the canine spontaneous breast cancer organoid model according to any one of claims 4 to 6, used in assessing the pathological characteristics, drug sensitivity, tumor marker expression, and degree of functional impairment of the breast cancer model.

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